Hallmarks of cystic fibrosis (CF) airway disease include bronchiectasis, inflammation by polymorphonuclear neutrophils (PMNs) from blood, obstruction by mucus, and infection by bacteria. In landmark studies, the AREST CF consortium showed that first signs of bronchiectasis in CF children occur in small airways and coincide with PMN recruitment and high activity of neutrophil elastase (NE) in the airway fluid. Recently, we showed that a large fraction of airway PMNs in chronic CF disease release NE while viable, as part of a reprogramming process that includes metabolic activation, caspase-1 activity and hyperexocytosis. Here, we show that similar hyperexocytosis occurs in airway PMNs from CF children. In addition, we introduce a new model of human small airway in which naive blood PMNs exposed to apical CF airway fluid in vitro are induced to transmigrate and become hyperexocytic, recapitulating features of CF airway PMNs in vivo. Our long-term goal is to develop therapies aimed at correcting PMN dysfunction, ideally via targeted intervention in early CF disease. As a step toward this objective, we will conduct a mechanistic study of PMN function in early CF leveraging: (i) our renewed understanding of PMN dysfunction in CF; (ii) our new in vitro model, and (iii) the unique AREST-CF study about to be initiated in the Netherlands. This prospective cohort is modeled after the Australian AREST-CF study, including computed tomography (CT) and airway fluid analysis at 12, 36 and 60 months to closely follow the onset and progression of early CF disease. Our overall hypothesis is that early CF disease coincides with airway PMN reprogramming. Our aims are to: AIM 1- Determine the extent of PMN reprogramming in early CF disease, in vivo. Hypothesis 1A: Airways in CF children contain reprogrammed PMNs. Hypothesis 1B: Progression of airway disease measured clinically in CF children associates with higher burden of reprogrammed PMNs. AIM 2- Identify mechanisms of PMN reprogramming by CF airway fluid, in vitro. Hypothesis 2A: Airway fluid from CF children induces PMN reprogramming in our model. Hypothesis 2B: Modulation of functional pathways in reprogrammed airway PMNs can inhibit their dysfunction and associated disease burden. To leverage the extraordinary research opportunity afforded by the parent study, we assembled a group of CF researchers with multidisciplinary expertise. This proposed ancillary study is highly time-sensitive, since the parent study will start shortly, and high-content assays included here (e.g., flow cytometry, image cytometry, multiplexed qPCR, targeted mass spectrometry) require immediate processing of patient samples. We expect this study to yield better mechanistic understanding of early CF disease and strong proof- of-concept data on new targeted therapies to help prevent the vicious cycle of CF airway inflammation.